PROJECT SUMMARY ? PROJECT 1 Oncolytic herpes simplex virus (oHSV) vectors offer considerable promise in the treatment of Glioblastoma Multiforme (GBM). In the current funding period we designed a new series of HSV vectors capable of safe but efficient tumor destruction through (i) full vector retargeting to prevalent human GBM receptors (EGFR/EGFRvIII) (vector KGNE), (ii) cellular miR-124 blockade of vector replication in normal brain (KGN-4:T124: our base vector), or (iii) a combination of these features (KGNE-4:T124). Further arming of the combination vector with a matrix metalloproteinase (KGNE-4:T124-MMP9) enhanced vector spread and oncolysis in animal models. However, vector replication in tumors can still be limited by Natural Killer (NK) cells that normally protect the host from HSV infection. Projects 2 and 4 have recently demonstrated that interference with NK cell activation and killing of virus-infected cells enhances GBM treatment by allowing more robust and broader oncolytic virus spread. We propose to validate and extend these findings by comparing the impact of vector transgene-mediated NK blockade on the oncolytic activities of the two base vectors of this PPG, our KGN-4:T124 (strain KOS) and vector rQNestin34.5 (strain F) from Project 2, in three distinct syngeneic mouse models of GBM. Because these base vectors have different genetic backgrounds and are controlled by different safety mechanisms, their comparison in these experiments will be important for choosing the most effective backbone and arming genes for future trials. We will exploit two HSV-permissive mouse glioma cell lines, CT2A and GL261N4, to generate brain tumors in immunocompetent mice by tumor cell injection. In addition, we have an established collaboration with Eric Holland (U. of Washington) to verify key results in his genetically induced mouse models of GBM based on the RCAS/tv-a system. In Aim 1, we will establish base-line tumor treatment efficacies for KGN-4:T124 versus rQNestin34.5. We will perform tumor-treatment dose-response analyses, and characterize and compare the effects of the two vectors on the composition of the tumor microenvironment (TME). In Aim 2, we will take advantage of these base-line data to evaluate the effect of NK cell antagonism on tumor therapy. In collaboration with Projects 2 and 4, we will determine the effects of vector arming with genes encoding soluble forms of HCMV UL141 and/or PRV glycoprotein D that reduce the expression of glioma cell ligands recognized by NK cells. In Aim 3, we will express the Fc?-binding ectodomains of two other HCMV-derived proteins, gp34 and gp68, that are known to inhibit NK cell activation. In collaborative experiments with Project 4, we will test the hypothesis that sgp34 will inhibit classical ADCC (antibody-dependent cellular cytotoxicity), enhance vector replication and tumor virolysis while sgp34 and/or sgp68 will interfere with Fc bridging cellular cytotoxicity (FcBCC). We will also provide TIMP-3 expression vectors to Project 3 to inhibit Notch signaling among macrophages and thereby reduce macrophage recognition of infected tumor cells and prolong oHSV persistence.